1. Technical Field
The subject invention relates to .beta.-casein expressing constructs which have significant stability when introduced into host cells. These constructs, for purposes of the present invention, have been designated as pRAB-84-69 and pRSB-14.
2. Background Information
Stability of a plasmid in an induced cell culture is influenced by the external physical culture conditions as well as the internal environment of the cell. There are several external factors that may have an effect on plasmid stability. For example, it has been shown that a lack of carbon, nitrogen, phosphate and minerals can negatively affect plasmid stability (Godwin et al., J. Gen. Microbiol. 111:201-10 (1979)). Use of a richer media may therefore alleviate plasmid loss in some cases.
The physiology of the host strain is another factor which has an important effect on plasmid stability, as the same plasmid can show different rates of loss depending on the host (Caulcott et al., J. Gen. Microbiology 133:1881-89 (1987)). Some plasmids are inherently unstable due to improper replication and segregation. Unstable segregation in the earlier stages of the culture gives a growth disadvantage to the cells harboring the plasmid, consequently leading to an increased proportion of cells without the plasmid. Maintenance of the plasmid in the culture also requires optimum levels of selective pressure on the culture to ensure that only cells harboring the plasmid with the selectable marker survive.
In addition to the above mentioned factors, the absence or presence of specific sequences may also affect plasmid stability. Chiang and Bremmer studied the stability of plasmid pBR322 and its tet, bla and rom derivatives (Chiang et al., Plasmid 20:207-220 (1988)). They reported that transcription of tet sequences present on pBR322 affects cell viability and may reduce plasmid stability. In other instances, the presence of certain sequences such as the par stability locus has been shown to stabilize plasmids with segregational instability (Austin et al., Plasmid 20:1-9 (1988)). Furthermore, the size of the inserted DNA and the "act of introducing" foreign DNA are also known to effect plasmid stability. Stability is negatively affected by inserts over 8 kb in size and has been attributed to replication fidelity, segregation or low copy number (Warnes et al., Plasmid 16:116-23 (1986)).
Such instability prevents large-scale or efficient production of the protein encoded by the nucleotide sequences present in the plasmid. Thus, such instability may have quite a large impact on the production level of the protein as well as on the development of diagnostic and therapeutic products. Additionally, genetic instability of the plasmid is important with respect to the receipt of regulatory approval for a commercial process.
The present constructs, derived from pRJB36, are quite stable and therefore permit the expression of large quantities of the desired end product. For example, the constructs may be used in the expression of a protein such as recombinant human .beta.-casein. Thus, the present constructs overcome many of the disadvantages associated with known constructs such as pRJB36.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.